The present invention relates to ciliary neurotrophic factor (CNTF) and CNTF-related polypeptides useful for the treatment of neurological diseases, obesity and other diseases or disorders.
CNTF is a protein that is required for the survival of embryonic chick ciliary ganglion neurons in vitro (Manthorpe et al., 1980, J. Neurochem. 34:69-75). The ciliary ganglion is anatomically located within the orbital cavity, lying between the lateral rectus and the sheath of the optic nerve; it receives parasympathetic nerve fibers from the oculomotor nerve which innervates the ciliary muscle and sphincter pupillae.
Over the past decade, a number of biological effects have been ascribed to CNTF in addition to its ability to support the survival of ciliary ganglion neurons. CNTF is believed to induce the differentiation of bipotential glial progenitor cells in the perinatal rat optic nerve and brain (Hughes et al., 1988, Nature 335:70-73). Furthermore, it has been observed to promote the survival of embryonic chick dorsal root ganglion sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39 46). In addition, CNTF supports the survival and differentiation of motor neurons, hippocampal neurons and presympathetic spinal cord neurons (Sendtner, et al., 1990, Nature 345: 440-441; Ip, et al. 1991, J. Neurosci. 11:3124-3134; Blottner, et al. 1989, Neurosci. Lett. 105:316 320).
CNTF has been cloned and synthesized in bacterial expression systems, as described by Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and in International Publication No. WO 91/04316, published on Apr. 4, 1991, both of which are incorporated by reference in their entirety herein.
In addition to human CNTF, the corresponding rat (Stxc3x6ckli et al., 1989, Nature 342:920-923), and rabbit (Lin et al., 1989, J. Biol. Chem. 265:8942-8947) genes have been cloned and found to encode a protein of 200 amino acids, which share about 80% sequence identity with the human gene. Both the human and rat recombinant proteins have been expressed at exceptionally high levels (up to 70% of total protein) and purified to near homogeneity.
Despite their structural and functional similarity, recombinant human and rat CNTF differ in several respects. The biological activity of recombinant rat CNTF in supporting survival and neurite outgrowth from embryonic chick ciliary neurons in culture is four times better than that of recombinant human CNTF (rHCNTF) (Masiakowski et al., 1991, J. Neurochem. 57:1003-1012). Further, rat CNTF has a higher affinity for the human CNTF receptor than does human CNTF (Davis et al., 1991, Science 253: 59-63).
A surprising difference in the physical properties of human and rat CNTF, which are identical in size, is their different mobility on SDS gels. This difference in behavior suggests the presence of an unusual structural feature in one of the two molecules that persists even in the denatured state (Masiakowski et al., 1991, J. Neurochem. 57:1003 1012).
To better understand the physical, biochemical and pharmacological properties of rHCNTF, applicants undertook rational mutagenesis of the human and rat CNTF genes based on the different biological and physical properties of their corresponding recombinant proteins (See Masiakowski, P., et al., 1991, J. Neurochem., 57:1003 1012). Mutagenesis by genetic engineering has been used extensively in order to elucidate the structural organization of functional domains of recombinant proteins. Several different approaches have been described in the literature for carrying out deletion or substitution mutagenesis. The most successful appear to be alanine scanning mutagenesis (Cunningham and Wells 1989, Science 244: 1081-1085) and homolog-scanning mutagenesis (Cunningham et al., 1989, Science 243:1330-1336). These approaches helped identify the receptor binding domains of growth hormone and create hybrid proteins with altered binding properties to their cognate receptors.
Applicants have found that the nature of the amino acid at position 63 could greatly enhance the affinity of human CNTF for soluble CNTFRxcex1 and its biological potency in vitro (Panayotatos, N., et al., J. Biol. Chem., 1993, 268:19000-19003; Panayotatos, N., et al., Biochemistry, 1994, 33: 5813-5818).
The CNTF receptor complex contains three proteins: a specificity determining xcex1 component that directly binds to CNTF, as well as two signal transducing xcex2 components (LIFRxcex2 and gp130) that cannot bind CNTF on their own, but are required to initiate signaling in response to CNTF. The xcex2 component of the CNTFR complex is more widely distributed throughout the body than the xcex1 component. The 3 components of the CNTFR complex are normally unassociated on the cell surface; 0CNTF induces the stepwise assembly of a complete receptor complex by first binding to CNTFR xcex1, then engaging gp130, and finally recruiting LIFRxcex2. When this final step in receptor assembly occurs (heterodimerization of the xcex2 components), intracellular signaling is initiated by activating non-receptor tyrosine kinases (JAK kinases) associated with the xcex2 components. JAK kinases respond by phosphorylating each other and also tyrosine residues on the receptor cytoplasmic domains, creating phosphotyrosine docking sites for the Src homology 2 domains of STAT proteins. After their phosphorylation, bound STAT proteins dissociate from the receptor, dimerize, arid translocate to the nucleus where they bind DNA and activate transcription (reviews: Frank, D. and Greenberg, M. (1996) Perspectives on Developmental Neurobiology 4: 3-18; Stahl, N. and Yancopoulos, G. (1997) Growth factors and cytokines in health and disease 2B, 777-809). Axokine(trademark) (rHCNTF, C17A, Q63 Rxcex94C15) (xe2x80x9cAx-15xe2x80x9d) is a mutant CNTF molecule with improved physical and chemical properties, which retains the ability to interact with and activate the CNTF receptor. (Panayotatos, N., et al. (1993) J. Biol. Chem. 268: 19000-19003).
Leptin, the product of the ob gene, is secreted by adipocytes and functions as a peripheral signal to the brain to regulate food intake and energy metabolism (Zhang, et al. (1994) Nature 372: 425-431). Interestingly, leptin receptor (OB-R), a single membrane-spanning receptor, has considerable sequence similarities to gp130 (Tartaglia, L., et al. (1995) Cell 83: 1263-1271). Both CNTF and leptin each signals through the JAK/STAT pathway (Baumann, H., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 8374-8378; Ghilardi, N., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 6231-6235). Systemic administration of both CNTF and leptin results in induction of tis-11 (Gloaguen, I., et al. (1997) Proc. Natl. Acad. Sci. USA 94: 6456-6461) and STAT3 (Vaisse, C., et al. (1996) Nature Gen. 14: 95-97) in the hypothalamic satiety center, indicating their roles in the regulation of body weight and feeding behavior. Indeed, in a clinical trial testing the use of CNTF for treating ALS, it was found that administration of CNTF to humans reduced food intake and resulted in weight loss (Group, A. C. T. S. (1996) Neurology 46:1244-1249.).
An object of the present invention is to provide CNTF and CNTF-related proteins, collectively referred to herein as CNTF proteins, for the treatment of diseases or disorders including, but not limited to, obesity and diabetes.
A further object of the present invention is to provide a method for administering CNTF or CNTF-related proteins and maintaining biological activity. A preferred embodiment of this invention is the administration of CNTF or a CNTF related protein to the nasal or respiratory system of a mammal to produce an increase in the level of the protein in the systemic blood circulation of the mammal. A particularly preferred embodiment comprises the administration of the modified CNTF molecule, designated herein as AX-15, to the nasal passages of a patient for the treatment of obesity or diabetes.